Association between Dynamic Contrast-Enhanced MRI Parameters and Prognostic Factors in Patients with Primary Rectal Cancer

Background: To evaluate the association between perfusion parameters derived from dynamic contrast-enhanced magnetic resonance imaging (DCE-MRI) with prognostic factors in primary rectal cancer patients. Methods: A sample of 51 patients with pathologically proven rectal adenocarcinoma through surgery were retrospectively enrolled. All the patients underwent preoperative DCE-MRI including 3D-spoiled gradient echo. Two radiologists determined the tumor border after radiologic–pathologic correlation and drew regions of interest. The perfusion parameters, including the volume transfer constant (Ktrans), were calculated under the extended Toft model. The prognostic factors included TN stage, circumferential resection margin, extramural venous invasion, Kirsten-ras mutation, tumor size, carcinoembryonic antigen, and tumor differentiation. The association was assessed via correlation or t-test. For significant prognostic factors, receiver operating characteristic (ROC) curve analyses were performed to estimate the diagnostic predictive values. Results: Ktrans only showed a significant difference according to tumor differentiation, between the well-differentiated (n = 6) and moderately differentiated (n = 45) groups (0.127 ± 0.032, 0.084 ± 0.036, p = 0.036). The AUC was 0.838 (95% CI, 0.702–0.929), and the estimated accuracy, sensitivity, and specificity were 87%, 90%, and 60%, respectively. Conclusions: Ktrans showed a significant difference based on tumor differentiation, which may be conducive to prediction of prognosis in primary rectal cancer.


Introduction
Rectal cancer is ranked third among the most common causes of death associated with malignancy and is one of the cancers that require a personalized treatment approach [1]. Almost 70% of patients are over 65 years of age and the 5-year survival rates after surgical excision are known to be 85-95% for stage I and less than 10% for stage IV cancer [2]. At the time of diagnosis, liver metastases account for 15-25% of colorectal cancer patients, which is one of the causes of high mortality [3]. It is known that some colorectal cancer patients with liver metastases can be cured with liver resection; however, the rate has been reported to be less than 20-30%. Therefore, combined chemotherapy including targeted therapy, with surgery has been attempted to cure the disease [4]. Like other cancers, rectal cancer is caused by genetic alterations that alter normal cell function. Spontaneous mutations-such as KRAS, APC, and TP53-are known to contribute to the development and progression of diseases. Using this, targeted therapies that specifically target mutations, which have fewer side effects and are more effective than generalized cancer treatments, are used for cancer treatment [5]. These genetic factors as well as clinico-pathological factors such as tumor

Materials and Methods
This retrospective study was approved by our institutional review board and informed consent from individual patients was waived.

Patients and Patient Selection Criteria
Between September 2019 and September 2022, patients who had been proven to have rectal adenocarcinoma via colonoscopic biopsy and who satisfied our inclusion criteria were initially recruited for this study. The inclusion criteria were as follows: (1) patients who underwent rectal DCE-MRI with a 3-Tesla scanner before surgery; (2) patients who underwent subsequent surgical excision without nCRT; and (3) patients who had complete datasets including medical, laboratory, and histopathologic report. A total of 53 patients (33 men, 20 women) were eligible. Among them, 2 patients were excluded due to the following reasons: (1) a patient with suboptimal scan coverage (n = 1); (2) a patient whose MR image quality was not suitable for perfusion analysis (n = 1); and (3) a patient who had been proven to have mucinous adenocarcinoma via histopathologic study (n = 0). A total of 51 consecutive patients (31 men, 20 women; mean age, 69 years; range, 45-89 years) were thus included in the study. The case enrollment process is summarized in Figure 1. 0). A total of 51 consecutive patients (31 men, 20 women; mean age, 69 years; range, 45-89 years) were thus included in the study. The case enrollment process is summarized in Figure 1.

MRI Technique
MRI examinations were carried out on a 3-Tesla MR scanner (Achieva TX; Philips, Best, Netherlands) using a torso coil (SENSE; USA Instruments, Gainesville, FL, USA). Antiperistaltic agents were not used. The scan protocols were as follows: T2-weighted imaging in 3 planes and axial T1-weighted 3-dimensional spoiled gradient echo (fast-field echo) imaging before and after contrast media injection. After the intravenous injection of 0.1 mmol/kg or 0.2 mL/kg of contrast agent (gadoterate meglumine, Clariscan, GE healthcare, Chicago, IL, USA) at a rate of 3.0 mL/s, 30 mL of normal saline was flushed. The temporal resolution of the DCE sequence was 3.6 s, and dynamic data acquisition was repeated 70 times for full coverage of the rectal cancer. The details of the scan parameters are given in Table 1.

Perfusion Parameter Measurement
The perfusion analyses were performed on a workstation (ISP, version 10.0, Philips, Best, Netherlands) equipped with a perfusion analysis tool (MR permeability). The MR permeability tool adopts an arterial input function (AIF) that is based on a populationbased medium bi-exponential model. This function can be pre-selected according to the user selective injection duration as follows; short (less than 5 s), medium (between 5 and

MRI Technique
MRI examinations were carried out on a 3-Tesla MR scanner (Achieva TX; Philips, Best, Netherlands) using a torso coil (SENSE; USA Instruments, Gainesville, FL, USA). Antiperistaltic agents were not used. The scan protocols were as follows: T2-weighted imaging in 3 planes and axial T1-weighted 3-dimensional spoiled gradient echo (fast-field echo) imaging before and after contrast media injection. After the intravenous injection of 0.1 mmol/kg or 0.2 mL/kg of contrast agent (gadoterate meglumine, Clariscan, GE healthcare, Chicago, IL, USA) at a rate of 3.0 mL/s, 30 mL of normal saline was flushed. The temporal resolution of the DCE sequence was 3.6 s, and dynamic data acquisition was repeated 70 times for full coverage of the rectal cancer. The details of the scan parameters are given in Table 1.

Perfusion Parameter Measurement
The perfusion analyses were performed on a workstation (ISP, version 10.0, Philips, Best, Netherlands) equipped with a perfusion analysis tool (MR permeability). The MR permeability tool adopts an arterial input function (AIF) that is based on a population-based medium bi-exponential model. This function can be pre-selected according to the user selective injection duration as follows; short (less than 5 s), medium (between 5 and 10 s), and long (longer than 10 s) [28]. Two radiologists (with 18 years and 2 years of experience in interpreting rectal MRIs, respectively) read all the MR images for each patient. After determining the tumor border, the junior radiologist placed the region of interest (ROI) along the tumor border for each consecutive post-contrast T1WI image to cover the whole tumor volume [29]. The perfusion parameters were automatically calculated using the extended Toft model [28,30]. The perfusion parameters were as follows: forward volume transfer constant (K trans ), fractional extracellular extravascular space (EES) volume (v e ), plasma volume fraction (v p ), and revere volume transfer constant (k ep ). The K trans was defined as the influx from the blood plasma to the EES. The k ep was defined as the efflux from the EES to the blood plasma. The volume fractions of EES and blood plasma were represented as v e and v p , respectively. The mean values were calculated as a representative by averaging each measured perfusion parameter values for the entire tumor [30].

Reference Standard for Prognostic Factors
Laboratory results, including CEA levels, were taken from patients' medical records. Pathologic examinations were conducted by a dedicated pathologist who had 10 years of clinical experience in accordance with the TNM staging system [6]. Tumor differentiation grading was divided according to the 2019 WHO grading system based on the percentage of glandular formation throughout the tumor, with cut-offs of >95%, 50-95%, and <50% indicating well, moderately, and poorly differentiated, respectively [31].
Existence of EMVI, circumferential resection margin (CRM) status, and immunohistochemical results including KRAS mutation, were also reviewed [6,7,10]. EMVI is the presence of tumor cells in the veins, beyond the proper muscle layer of the rectal wall. EMVI can provide a pathway for hematogenous dissemination of tumor cells, and therefore, is an independent risk factor for disease relapse and metastasis [32]. KRAS mutation, which is found in 30-50% of colorectal cancer patients, has been demonstrated to be a predictive biomarker of resistance to anti-epidermal growth factor receptor (EGFR) therapy. It is well known that KRAS mutation is associated with poor response to anti-EGFR therapy [10,33].

Statistical Analysis
The association between the perfusion parameters and the prognostic factors was assessed using a correlation test for continuous data between prognostic factors or t-tests for categorical data. In the case of a significant association, receiver operating characteristic (ROC) curve analyses were performed. An area under the ROC curve (AUC) was calculated and considered the diagnostic performance. The sensitivity, specificity, and accuracy were estimated under the optimal cut-off value. Correlation analysis was also performed to study the relationship between perfusion parameters and measurable prognostic factors. The MedCalc software for Windows (MedCalc Software version 20.113, Mariakerke, Belgium) was used for all the statistical analyses, and p-values less than 0.05 were considered significant.

Patient Demographics
The mean duration between MRI and surgery was 9 days (range, 1-36 days). The average tumor size was 4.66 cm (range, 0.6-11 cm), and the average CEA level was 10.98 ng/mL (0.3-280 ng/mL). The location of the tumor was in the upper (n = 15), mid (n = 20), or lower rectum (n = 16). The tumor differentiation was composed of moderately differentiated (n = 45) and well-differentiated tumors (n = 6). A pathologic EMVI was observed in eight patients. KRAS mutations were observed in 22 patients. The detailed demographic data, including the tumor and node stage, of the study population are given in Table 2. Patients with locally advanced rectal cancer (stage ≥ II) routinely underwent nCRT prior to surgical resection in our hospital. However, patients with colonic obstruction due to rectal cancer underwent surgery first without nCRT. Although it is controversial, 10 patients with good T3 (extramural growth < 5 mm) and node negative on MRI also underwent surgical resection first, then followed by adjuvant chemotherapy.
Curr. Oncol. 2023, 29, FOR PEER REVIEW 6 patients with good T3 (extramural growth < 5 mm) and node negative on MRI also underwent surgical resection first, then followed by adjuvant chemotherapy.

Comparison of Perfusion Parameters According to Prognostic Factors
Among the perfusion parameters and prognostic factors, only K trans and kep showed a significant difference by tumor differentiation-between the well-differentiated and moderately differentiated groups (K trans , 0.127 ± 0.032, 0.084 ± 0.036, p = 0.036; kep, 0.623 ± 0.252, 0.415 ± 0.151, p = 0.005, respectively) (Figures 2 and 3). Of the two perfusion parameters, only K trans showed a significant AUC (0.838, 95% CI, 0.702-0.929, p < 0.0001). Under the cutoff value of 0.130, the estimated maximum accuracy, sensitivity, and specificity were 87% (41/47), 90% (38/42), and 60% (3/5), respectively. On the other hand, kep showed an AUC of 0.758 (95% CI, 0.616-0.868, p = 0.08), which was not statistically significant.  In addition, the TN stage did not show significant differences for four perfusion parameters (p > 0.05). The EMVI positive (n = 8) did not show a significant difference compared with the EMVI negative (n = 43) for four perfusion parameters (p > 0.05). The KRAS positive (n = 22) did not show a significant difference compared with the KRAS negative (n = 29) for four perfusion parameters (p > 0.05). The CRM positive status (n = 8) did not show a significant difference compared with the CRM negative status (n = 43) for four perfusion parameters (p > 0.05), either. The statistical results are given in Table 3.
In a subgroup analysis, the tumor size was also significantly different between the well-differentiated and moderately differentiated groups (2.82 ± 1.78 and 4.72 ± 2.26, respectively, p = 0.0496). However, the CEA levels were not significantly different from each other (1.86 ± 0.47 and 7.90 ± 7.67, respectively, p = 0.2243). The results are summarized in Table 4. In addition, the TN stage did not show significant differences for four perfusion rameters (p > 0.05). The EMVI positive (n = 8) did not show a significant difference co pared with the EMVI negative (n = 43) for four perfusion parameters (p > 0.05). The KR positive (n = 22) did not show a significant difference compared with the KRAS nega (n = 29) for four perfusion parameters (p > 0.05). The CRM positive status (n = 8) did show a significant difference compared with the CRM negative status (n = 43) for f perfusion parameters (p > 0.05), either. The statistical results are given in Table 3.     In terms of correlation coefficients, there was no significant correlation between the tumor size and K trans (r = −0.081, 95% CI, −0.351-0.202, p = 0.585) or between the CEA level and K trans (r = −0.143, 95% CI, −0.408−0.144, p = 0.327).

Discussion
We assessed the association between MR perfusion parameters and various prognostic factors for primary rectal cancer. Our results showed that K trans only showed a significant difference according to tumor differentiation. Specifically, the value was higher in the well-differentiated group than in the moderately differentiated one. These results are contrary to those of previous studies [2,26]. Shen et al. reported that the K trans value was lower in the well-differentiated group (n = 6) than in the moderately differentiated one (n = 21) (0.182 ± 0.153, 0.280 ± 0.067, respectively, p = 0.004) [2]. However, the values between the moderately and poorly differentiated groups (n = 13) were not significantly different (0.284 ± 0.068). Another study by Li et al. also showed that the K trans value was higher in the poorly differentiated group (n = 41) than in the well-(n = 6) and moderately differentiated ones (n = 51) (0.92 ± 0.51, 0.53 ± 0.29, respectively, p < 0.001) [26].
The discrepancy between our observation and previous studies could be attributed to the difference in whole tumor coverage during the measurement process. A previous study only selected a representative image in each plane with no less than 1 cm 2 to measure perfusion parameters [2]. Another previous study manually drew the ROI to cover the entire tumor while excluding internal necrosis and cystic portions [26].
From the viewpoint of the tumor environment, mucin production and internal necrosis are frequently seen in rectal cancer and constitute tumor heterogeneity [34,35]. Contrary to previous studies, the consecutive image slices bearing whole tumor portions were covered to measure the perfusion parameters of the entire tumor while including internal necrosis and cystic portions containing mucin in our study [26]. Based on our experience, the larger the tumor, the more heterogeneous the tumor environment. In fact, the tumor size was larger in the moderately differentiated group than in the well-differentiated one in our study (4.72 ± 2.26 cm, 2.82 ± 1.78 cm, respectively). Thus, the heterogeneous tumor environment would be more reflected in the present study than in previous ones in which only pure solid tumor portions were included.
We acknowledge that the tumor differentiation by itself affects the neo-vascularization of the tumor; that is, newly developed fragile and impaired vessels are more frequently seen in poorly differentiated tumors than in well-differentiated ones. As a result, higher permeability (a higher K trans value) of the tumor is supposed to be observed in the more disorganized tumor environment with poorly differentiated tumor cells [2].
In terms of the therapeutic response to nCRT in locally advanced rectal cancer, K trans showed a significant decrease in the responder (tumor-downstaged group), whereas it was unchanged in poor responders (tumor-non-downstaged group) (in responders, from 1.24 ± 0.53 to 0.76 ± 0.45, p = 0.0007; in non-responders, from 1.02 ± 0.53 to 0.87 ± 0.48, p = 0.2358) [6]. However, the mean K trans values of the pre-CRT MRIs were not different from each other (p = 0.1393).
Although KRAS mutation status and EMVI did not show significant differences between the other perfusion parameters in our study, those factors showed a potential benefit in the literature [25,[36][37][38]. KRAS mutation is an independent prognostic factor and is known to be resistant to chemotherapy in rectal cancer [36]. Thus, patients with the wild type (non-mutated KRAS) show a favorable response to chemotherapy compared to patients with the KRAS mutation. Our results correspond well with a previous study. Yeo et al. also observed that K trans in a KRAS-mutated group showed no significant difference compared with a wild-type group (0.123 ± 0.032, 0.100 ± 0.039, respectively, p = 0.06) [17].
EMVI is also known to be a prognostic factor in rectal cancer. A previous study revealed that K trans was higher in the EMVI-positive group than in the EMVI-negative group (1.08 ± 0.946, 0.542 ± 0.636, respectively, p = 0.02) [25]. In addition, K trans combined with Ve showed a moderate accuracy (AUC, 0.715 in the validation cohort) for the preoperative prediction of EMVI in rectal cancer. Another study also observed that a MRdetected EMVI (mrEMVI)-positive group showed a higher median K trans value than a mrEMVI-negative group (0.74, 0.39, respectively, p < 0.01), and the AUC was 0.779 [37]. The discrepancy between those results and ours could be explained by the composition of the study population. In our study, most of the patients with T3 stage showed the EMVI negative except for three patients with bad T3 (extramural growth > 15 mm). Thus, no significant difference in K trans could be attributed to the relatively small number of the EMVI positive (n = 8) compared to a large number of the EMVI positive in T3 or T4 stage in those studies (n = 28, n = 24) [25,37].
Regarding the future perspectives of quantitative imaging modalities including DCE-MRI, diffusion weighted imaging, texture analyses, and radiomics on rectal cancer, much evidence has been collected to support consensus statements beyond the expert opinions [38]. Particularly, the amount of research into MRI-based radiomics in locally advanced rectal cancer has increased. Several studies have shown that MRI radiomics are useful in predicting tumor response after nCRT [39][40][41]. In addition, radiomics can be combined with emerging artificial intelligence, so-called deep learning to create a new field of predicting treatment response and prognosis.
Several limitations should be acknowledged in this study. First, the small study population of the well-differentiated group should be considered. However, well-differentiated tumors are known to constitute 10% of rectal adenocarcinomas, and thus they were enrolled in this study at a reasonable rate. In contrast, poorly differentiated tumors, which are known to constitute 15% of adenocarcinomas, were absent in the present study. The reasons are pathological sampling bias associated with tumor heterogeneity and the inherent subjective nature of the WHO grading system that our pathologist followed [31]. Second, inter-observer agreement in the measurements of perfusion parameters were not evaluated because the data from the whole tumor volume were known to be most reproducible among various measurement methods including single ROI or several ROIs covering a small solid tumor portion in a quantitative analysis in rectal cancer [42]. Third, the range of reproducibility according to different vendors was known to be within 20%, which is an inherent limitation in DCE-MRI [43,44]. Last, external validation could not be conducted because acquisition of DCE-MRI is considered as a research tool and not adopted routinely, thus it is very limited in other hospitals even in the tertiary referral hospitals.

Conclusions
In conclusion, K trans showed a significant difference according to tumor differentiation, which may be conducive to the prediction of prognosis in primary rectal cancer. Informed Consent Statement: Patient consent was waived due to extremely low risk to patients associated with this retrospective study.

Data Availability Statement:
The data presented in this study are available on request from the corresponding author. The data are not publicly available due to patients' privacy.

Conflicts of Interest:
The authors declare no conflict of interest.